Abstract Despite effective treatments for HIV, the presence of viral reservoirs has prevented the development of a cure for HIV infection. Although most reservoir work has focused on CD4+ T cells, these are not the primary infected cells in the CNS instead long-lived macrophages and microglia are infected. Furthermore, many of the anti-retroviral treatments do not penetrate the brain. In addition, drug abuse is frequently co-morbid with HIV infection and affects CNS virus and disease. Studying the state of virus in the brain, as well as doing controlled experiments with drugs of abuse, cannot be performed in humans. Here we will use the highly relevant SIV- infected macaque model treated with an effective anti-retroviral treatment regimen in three groups: those receiving the stimulant methamphetamine, those receiving the opiate morphine and those receiving no drugs of abuse. Our experimental design will test the hypothesis that the brain is a reservoir under controlled treatment regimens and determine the effects drugs of abuse have on such a reservoir. In parallel, we will examine a known reservoir (lymph node CD4+ T cells), both as a comparison and to illuminate additional effects of drugs of abuse on reservoirs. Specific Aim 1 will examine the effects of drugs of abuse on modulating the effectiveness of antiretroviral treatment in SIV infected macaques. Innovative aspects include our hypothesis based mechanistic studies examining changes in cellular markers that may impact infection and tissue migration, and our analyses of intracellular antiretroviral concentrations uniquely performed here in brain cells as well as in lymphoid cells in the setting of drugs of abuse. Specific Aim 2 will utilize a series of state of the art assays to examine the hypotheses that under suppressive anti-retroviral treatment, the brain is indeed a viral reservoir for SIV and substance abuse increases the size of this reservoir. We will utilize PCR detection, viral outgrowth in indicator cells and the highly sensitive TILDA assays to examine virus in microglia/macrophages purified from the brains of the animals in Aim 1. To ensure a definite result with high sensitivity and specificity, we will adoptively transfer infection to nave monkeys as a new platinum standard for reservoir detection to definitely address this question of the CNS as a reservoir and the effects of drugs of abuse. Such knowledge is crucial for devising strategies to cure HIV infection, including in drug abusers.